Jadzia Dax and other Trills portrayed in DS9 were distinctly different from how Trills had been depicted in the TNG episode "The Host". In DS9, the relationship between host and symbiont was described more as a truly symbiotic relationship rather than the symbiont dominating the host. Trills now having spots, rather than prosthetic make-up, was due to studio executives feeling that Jadzia Dax actress Terry Farrell was too attractive to cover her face up. (Cinefantastique, Vol. 23, No. 6, p. 21)

Another significant change was the relationship Ferengi had with Humans. The Ferengi on TNG had originally been intended to be a new adversary comparable to the Klingons in TOS, although the writers had quickly realized how ridiculous the Ferengi were as villains. In DS9, the Ferengi were mainly entrepreneurs and the Ferengi Alliance was a politically neutral economic power.

In addition to the visits to the mirror universe, the series also featured a number of episodes in which the character of Miles O'Brien was subjected to particular trauma. This became an in-joke among the DS9 writing staff, who called them "O'Brien Must Suffer" episodes and went to great lengths to produce at least one such episode per season. (Star Trek: Deep Space Nine Companion)

The show also focused on a wider array of uses and depictions of functions for holographic simulations (known as a holodeck in TOS and TNG but as a holosuite in DS9). In addition to many obvious activities (such as those referenced by Chief O'Brien and Julian Bashir) which were completely in keeping with holodeck usage on The Next Generation, the numerous applications of the holosuites on DS9 included them being used as: a recurring background for people to hang out in, in the form of a 1960s Las Vegas lounge (in numerous episodes); a weapons showroom (by Quark); and the location for a baseball game between teams assembled by Sisko versus Solok, a long-time rival Vulcan captain (in "Take Me Out to the Holosuite").

The show broke the "standard format" for Star Trek shows a number of times as well, with a direct, first-person narrative providing the commentary for the episode "In the Pale Moonlight", a retelling of a classic TOS episode from a different angle in "Trials and Tribble-ations", life in the racially segregated 1950s in "Far Beyond the Stars", and a reintroduction of the concept of "black ops" to the Star Trek universe with Section 31: "Inquisition". The show also broke with tradition – and with the two Star Trek series that followed it – by featuring a commanding officer as the star of the show at the rank of commander, rather than captain, for a significant portion of its run, until Sisko was eventually promoted to captain in "The Adversary".

Deep Space Nine was the first live-action Star Trek series to feature a fully animated sequence in its opening credits, as opposed to the simple flashcards accompanied by rapid flypasts of the Enterprise used for the opening sequences of both The Original Series and The Next Generation. Subsequent series Voyager also had a fully animated credit sequence.

The decision to set the series on a space station, rather than a starship, spawned when Brandon Tartikoff originally approached Rick Berman about the show, in 1991, and specifically said he wanted it to have a format that was new for Star Trek but was classicly western; if The Next Generation was Wagon Train in space, Deep Space Nine was to be The Rifleman in space – a man and his son coming to a dilapidated frontier town on the edge of known civilization. Berman brought this concept to Michael Piller, and together they set about creating a western in space. As Robert Hewitt Wolfe later explained, "We had the country doctor, and we had the barkeeper, and we had the sheriff and we had the mayor, we had it all, it was all there. We had the common man, Miles O'Brien, the Native American, Kira." Indeed, the producers initially discussed setting the show at a colony on an alien planet rather than on a space station. This idea was ultimately rejected because it was felt that it would involve too much location shooting, and because they felt that fans of Star Trek wanted to see storylines set primarily in space, not on a planet. (New Frontiers: The Story of Deep Space Nine, DS9 Season 2 DVD special features)

The change of venue to a space station was largely intended to differentiate DS9 from The Next Generation, because the producers felt that having two shows about a starship airing simultaneously would be unacceptable. As co-creator and executive producer Rick Berman later explained, "Because there were two years of overlap with The Next Generation, we could not create a show that took place on a spaceship. It just seemed ridiculous to have two shows and two casts of characters that were off going where no man has gone before. It was a land-based show, it was a show that in a sense was taking place on a space station. So it had to be an entirely different concept." (Deep Space Nine: A Bold Beginning, DS9 Season 1 DVD special features)

The decision to set the show on a fixed station rather than a traveling starship was also based upon a desire to look deeper into the actual workings of the Federation and to see how it dealt with the type of problems one wouldn't find in a show set upon a starship. Michael Piller felt that, by having the characters standing still, they would be forced to confront issues not usually applicable to people on a starship. Whereas on The Next Generation, issues raised each week could simply be forgotten about the following week as the ship visited somewhere else, on a space station, events couldn't be forgotten or left behind but instead had to have implications for the future. As Piller explained, "We didn't want to have another series of shows about space travel. We felt that there was an opportunity to really look deeper, more closely at the working of the Federation and the Star Trek universe by standing still. And by putting people on a space station where they would be forced to confront the kind of issues that people in space ships are not forced to confront. In a series that focuses on a starship, like the Enterprise, you live week by week. You never have to stay and deal with the issues that you've raised. But by focusing on a space station, you create a show about commitment. It's like the difference between a one-night stand and a marriage. On Deep Space Nine, whatever you decide has consequences the following week. So it's about taking responsibility for your decisions, the consequences of your acts." (Star Trek: Deep Space Nine Companion) Similarly, in 2002, Piller stated, "If you look at The Next Generation, it's really about movement. You don't ever stay in one place long enough to get to know anybody. Well Deep Space Nine is a show where everybody is forced to stay week after week, so each episode, each show, is fundamentally dealing with the people who have to learn that actions have consequences, and they have to live with the consequences of their actions on a weekly basis." (New Frontiers: The Story of Deep Space Nine, DS9 Season 2 DVD special features)

Setting the show in a fixed location meant that a large cast of recurring characters could be built up with relative ease; much more so than in The Original Series or The Next Generation before it, or Star Trek: Voyager, Star Trek: Enterprise or Star Trek: Discovery since. As Rick Berman, speaking in 2002, stated, "The show was land-based, but the benefit we got from that was that by staying in one place, it enabled us to create twenty or thirty secondary and recurring characters, which really enriched the show because of all the multi-layers of relationships that have existed over the years. It's a very character-driven show as a result, and I think that makes it quite unique." (Deep Space Nine: A Bold Beginning, DS9 Season 1 DVD special features)

The decision to set the show in a fixed location was regarded as a benefit by the series' staff writers. For example, Ira Steven Behr, speaking in 1996, commented, "We have certain advantages that I think no other Star Trek series has had, because we do have a base of operations that doesn't travel through space, which is the space station. Every story we do, the repercussions, the consequences don't disappear. It's not like the other shows where you have an adventure and then you zoom off into the great unknown. We are here, we have made a home, what we do has consequences. And I think we're able to do this mosaic, this fabric of life in the future, which I like." Similarly, Robert Hewitt Wolfe, speaking in 2002, stated, "I think if Next Generation and The Original Series were about going out there and discovering new things about other races, Deep Space Nine is about staying in one place and discovering new things about ourselves. Not that we didn't go out there and discover things, but we had the same characters, we didn't change location every week. Sisko couldn't just solve a problem and sail off into the sunset, and never have to go back to that place again. That place was always there, and that problem could always come back to haunt him. So, in a lot of ways, it was a more complex show." (New Frontiers: The Story of Deep Space Nine, DS9 Season 2 DVD special features)

The series was designed to have more interpersonal conflict than its predecessors, while still staying true to the universe that Gene Roddenberry had created. Rick Berman commented, "[Deep Space 9]'s an alien space station that doesn't work the way they want it to, and that in itself created a lot of conflict. At the same, our core characters are Starfleet officers; Sisko, O'Brien, the doctor and Dax in no way vary from The Next Generation in terms of the lack of conflict among themselves. That was a rule we had to follow." (Captains' Logs Supplemental - The Unauthorized Guide to the New Trek Voyages, p. 5) Berman also commented, "What we wanted to do was something that was almost paradoxical – bring conflict but not break Gene's rules. They still play paramount importance in what we're doing. We created an environment where Starfleet officers were in a location that they weren't happy about being in, and they were in a location where the people who lived there weren't all that happy about them being there." (Captains' Logs Supplemental - The Unauthorized Guide to the New Trek Voyages, p. 8)

The show's main cast was intentionally assembled to create conflict (Quark and Odo, Kira and Sisko, etc.), so as to contrast the relatively tranquil atmosphere aboard Federation starships. This was another very specific decision taken by the producers. Gene Roddenberry's golden rule was that there was to be no conflict among Starfleet characters, so the producers decided to introduce non-Starfleet characters so conflict could come from within the show rather than always coming from outside (as it did on TNG). Rick Berman recalled, "We [….] created a situation where we had people who were members of our core group who were not Starfleet: the security shapeshifter Odo; the Bajoran Major, Kira; the bartender, Quark. A group of our integral people are not Starfleet officers, and the ones that are Starfleet officers aren't crazy about where they are, so we have a lot of frustration and conflict." (Captains' Logs Supplemental - The Unauthorized Guide to the New Trek Voyages, p. 8) Writer Joe Menosky explained, "You can see right away they're not the perfectly engineered Humans of TNG. They seem more real. I don't know if that makes them as attractive to viewers or not. But they are really different, and they represent a different way to tell a story. And it was definitely a conscious choice to create that potential for conflict." Similarly, Berman stated, "Viewers didn't see that group of loving family members that existed on the first two Star Trek shows." (Star Trek: Deep Space Nine Companion) Michael Piller also commented on this somewhat controversial aspect of the show; "One of the primary goals of the development process was to come up with a show that had more inherent conflict than The Next Generation. In order to do that, you have to understand that Gene Roddenberry had a very specific vision for humanity in the 24th century. What that meant for The Next Generation was that everybody gets along remarkably well on the Enterprise. There's very little room for interpersonal conflict between those people. In this series, we set out to create a situation that would provide natural conflict. We've populated the show with several aliens, primarily Bajorans, as we are stationed on the edge of the Bajoran star system. And the Bajorans are very different people than we are. They are people who are very spiritual and mystical and have a whole different way of looking at life than the 24th century humanist views which many of our Starfleet people will have. So immediately, there are conflicts. And then there's additional aliens from elsewhere who are thrown into the mix. So, as regular characters, not all the people are Starfleet, not all the people are Human, and as a result, you have this continuing conflict, because people who come from different places, honorable, noble people, will naturally have conflicts." (Deep Space Nine: A Bold Beginning, DS9 Season 1 DVD special features)

Unlike with TOS and TNG, Gene Roddenberry wasn't directly involved in conceptualizing DS9. Regarding Roddenberry's involvement in the series, Rick Berman stated, "Michael and I discussed it with Gene when we were still in the early stages, but never anything conceptual." "We never got a chance to discuss it (the concept) with Gene. By the time we had it to the point that it was discussable, he was in pretty bad shape and not really in the condition that it would have been wise to discuss it with him. On two specific occasions I was with him at his house and we tried to bring it up, but it wasn't really appropriate." (Captains' Logs: The Unauthorized Complete Trek Voyages, p. 328) Director Paul Lynch remarked, "My gut feeling is that Gene would be jumping up and down. This is definitely a different take on what Gene spawned, but I think he would love it [….] While it's quite different, Deep Space Nine is also, in many ways, quite the same. All of Gene's moral requirements are upheld in this show. If we've done anything, we've expanded on what Gene created." (The Official Star Trek: Deep Space Nine Magazine issue 1, pp. 10 & 12)

Initially, Berman and Piller were at a loss for a title for the series and toyed with calling the series "The Final Frontier". During further development, the station was temporarily dubbed "Deep Space Nine", which not only stuck permanently as the name of the station, but also the title of the show itself. Despite this, the two co-creators were reportedly dissatisfied with the name. (Trek: Deep Space Nine: The Unauthorized Story, pp. 17-18)

Miles O'Brien was brought aboard DS9 and made a part of the space station's senior staff because the producers felt that Colm Meaney was too talented an actor to confine his character to a transporter room. Additionally, they hoped the TNG crossover would help boost the new series' ratings.

In the Star Trek: Deep Space Nine Companion, Michael Piller explained the rationale behind each of the principal cast members, why each character was chosen, and what each one was to bring to the mix;

Jadzia Dax: "The Trill is a great race. They had some interesting ramifications on TNG. A Trill character would provide great potential for dichotomy and paradox."

Odo: "We knew that we needed some kind of Data/Spock character who looks at the world from the outside in. And the idea that an alien entity would have to find some way to pass as Human was fascinating, and seemed to give us an avenue into the kind of 'complexion of humanity' stories that we wanted to tell."

Quark: "A Ferengi would provide the show with instant humor and built-in conflict. I saw Quark as the bartender who is a constant thorn in the side of law and order, but who has a sense of humor about it. He'd be someone who could obviously throw lots of story dynamics into play."

Julian Bashir: "We decided to create a flawed character. He'd have to be brought down to size in order to grow. And we wrote him as kind of a jerk for much of the first season."

Miles O'Brien: "After we decided we were bringing him over to the new show, we thought, 'How do we use him?' We'd already decided to focus on Bajor, with this long backstory, establishing his bitterness towards the Cardassians, so it worked very nicely together."

Kira Nerys: "We liked the idea of having somebody working with the commander of the station who would be a thorn in his side, who would represent a different point of view. We knew we'd get conflict and interesting dynamics between the two characters."

Benjamin Sisko: "Every hero needs a journey. You want to take your leading man on a quest where he has to overcome personal issues as well as whatever space stuff happens to be out there. The idea of a man who is broken and who begins to repair himself is always a great beginning for drama."

The first officer aboard DS9 would have been Ro Laren, but she was replaced by Kira Nerys (Nana Visitor) because Michelle Forbes did not want to commit to a six-year contract working on DS9. Indeed, the reason the producers had decided to set the show on Bajor in the first place was because of Ro.

During preproduction for the series, the producers were especially keen to ensure that the aesthetic of the show was very different from anything yet seen in the Star Trek universe. For example, Director of Photography Marvin Rush said the producers told him that they wanted "a darker, more sinister place" than the Enterprise-D. Rush himself described the final look as "dark and shadowy." Similarly, Herman Zimmerman said, "The marching orders for the station were to make it bizarre." Finally, Supervising Producer David Livingston summed up the differences between DS9 and TNG by comparing the Enterprise's bridge with Deep Space 9's Ops; "The bridge is a very easy set to shoot. It's a three-wall open set with a lot of room, big and cavernous. Ops, on the other hand, is a multilevel set with a lot of cramped areas and very contrasty lighting. It's more interesting visually." (Star Trek: Deep Space Nine Companion) As Colm Meaney elaborated, "Because it was an alien space station, it gives the whole thing a very different feel I think to Next Generation or the original show, where you have the Enterprise, which is this very perfect environment. This is much more kind of dark and eerie, and also nothing works, the whole thing is a terrible mess." (Deep Space Nine Scrapbook: Year One, DS9 Season 1 DVD special features)

From the very beginning, DS9's darker aesthetic, more antagonistic characters and less utopian setting were somewhat controversial amongst die-hard fans of Gene Roddenberry's universe. As Ira Steven Behr, speaking in 1996 (about halfway through the show's seven-year run), stated, "At the beginning of Deep Space Nine's life, there was feelings that this was not a show that Gene would approve of by some of the fans, feeling that, you know, we had gone away from the image of the future as a paradise, that we had much more conflicts between our people, life isn't always great. But I think Gene, just by his very nature as a creative individual, as a writer, as a forward-thinking person, knows that any franchise has to move forward like a shark, or it dies. And I think he would understand what we're doing, and I think he would like what we're doing, and I think we're in the pocket of the Star Trek universe, and we try to push the envelope. And I see nothing wrong with that, and I have a hard time believing that Gene would see anything wrong with that." (New Frontiers: The Story of Deep Space Nine, DS9 Season 2 DVD special features) The sense that DS9 was too "dark" to be a Star Trek show only increased over the years, with episodes such as "Nor the Battle to the Strong", "In the Pale Moonlight" and "The Siege of AR-558", and topics such as Section 31 charting territory never before seen on a Star Trek show, and creating a great deal of controversy amongst fans of both The Original Series and The Next Generation.

Robert Hewitt Wolfe recalled that Sisko holding the rank of commander led to unfavorable comparisons to the other series. "Whenever people would do articles about Star Trek they would talk about the three captains: Kirk, Picard, and Janeway." The decision to promote Sisko to captain was prompted by the producers feeling that he deserved the higher rank as much as the other lead characters. (Star Trek: Deep Space Nine Companion, p. 253)

Identifying one way in which he believed DS9 differed from TNG, Colm Meaney stated, "On Next Generation they were dealing with more philosophical ponderings where we on DS9 tend to deal with more hands-on immediate crises that I think of as more resonant with the problems we have in the world today [….] I think that's probably the single main difference. We connect more with contemporary issues, issues relevant to the 1990s, than did Next Generation." ("Mr. Goodwrench", Star Trek: Communicator issue 105, p. 20)

Due to the non-episodic nature of DS9, much of the series was easily lost on the casual viewer. Many also believe that the changing television landscape contributed to DS9's ratings trouble, as local TV stations which had aired TNG in prime time became WB and UPN affiliates and pushed syndicated programming to the margins. Subsequent Star Trek shows Star Trek: Voyager and Star Trek: Enterprise had network support from UPN and a guaranteed time slot. DS9 was also the only series to run opposite another Star Trek show (first The Next Generation, then Voyager) for the entirety of its run (the first twelve episodes of the third season aired without another series on). Additionally, certain markets, notably in the UK, would only play one Star Trek series, in its entirety, at a time. Thus, events alluded to in The Next Generation or Voyager that happened in Deep Space Nine took months to "sync up."

Despite these problems, Deep Space Nine remained a fan-favorite series throughout its seven-year run, with reviewers consistently lauding the series for its bold shift in tone from The Next Generation. Most notable among such changes was the concept of inter-personal conflict – something which Gene Roddenberry himself was said to have forbidden.

"I'd like us to be remembered as the Trek series that dared to be different. We took chances in a franchise that has every reason to play it safe and spoon-feed the same old thing to the audience week after week. We challenged the characters, the audience, and the Star Trek universe itself. Sometimes we failed (sometimes spectacularly) but we never stopped trying to push the show into new directions."

Robert Hewitt Wolfe remarked, "The truth of DS9 is, we had a great ensemble cast. Michael Piller created all these terrific characters [with the exception of Worf]." ("Flashback: The Way of the Warrior", Star Trek Magazine issue 127)

There was also a rivalry with another popular and critically acclaimed television series, Babylon 5, created and produced by J. Michael Straczynski for Warner Bros. The two productions, which ran largely concurrently, were observed to be so similar that Babylon 5 fans accused Paramount, to whom Straczynski had previously pitched his series, of plagiarism. Considering how fellow Star Trek alumni like Walter Koenig and Andreas Katsulas had major roles in the rival series, Majel Barrett-Roddenberry agreed to a guest appearance in Babylon 5 as a gesture of goodwill to encourage a reconciliation between the two sets of fans.